Proteinases and mobilization of the entomotoxic protein Canatoxin, during germination of Canavalia ensiformis seeds.
Demartini, D.R.(1); Oliveira, T. F.(1).; Juliano, L.(2); Juliano, M. A.(2); Wlodawer, A.(3); Carlini, C.R(1).
Laboratory of Toxic Proteins – Department of Biophysics – UFRGS. Porto Alegre, Brazil (1); Department of Biophysics – EPM – UNIFESP. São Paulo, Brazil (2); Macromolecular Crystallography Laboratory – NCI. Frederick, USA(3).
Seeds of Canavalia ensiformis are a rich source of proteins, In 1981,Carlini & Guimarães isolated a potent insecticidal toxin, Canatoxin (CNTX), an isoform of the seed ureases. Previous data suggested that insect cathepsin B-like (CB) and cathepsin D- like (CD) enzymes cleave CNTX in vivo and in vitro, to release a 10 kDa entomotoxic peptide. In order to elucidate the mobilization of CNTX during germination, several enzymes, including CD and CB, as well as a candidate sedolisin (SDL) are under study. After germination (1 to 20 days) freeze-dried jackbean seeds/seedlins were ground and aqueous extracts were assayed for enzymatic activity. The CB activity was evaluated at pH 5,6, using Z-Phe-Arg-MCA, in the presence/absence of E-64 (5uM). CD (inhibited by 10 uM pepstatin-A) and SDL (inhibited nM 20 uM tyrostatin) were assayed with Abz-AIAFFSRQ-EDDnp, pH 3.5. Partial purification of the SDL was achieved by applying the crude extract (4 days of germination) into a CM-Sepharose column. The pass-through fraction was chromatographed on Q-Sepharose, the activity being eluted with 300 mM NaCl. The fractions were checked for in vitro hydrolysis of CNTX. Biossays with the insect Dysdercus peruvianus and fractions containing peptides prepared from plant tissues indicated the presence of CNTX-derived entomotoxic(s) peptide(s) after 4 days of germination. The aim of the work is to check if CNTX is being hydrolyzed during germination, which enzyme(s) are involved, when it is happening, and if toxic peptides are released as a defense mechanism of the plant.
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